The present invention is related to a transgenic mouse expressing green fluorescent protein under the control of a human glial fibrillary protein promoter and to a method of determining the neurotoxicity of substances in vivo.
Neural tissue consists of neurons and supporting or glial cells. Glial cells outnumber neurons by about ten to one in the mammalian brain. Glial cells may be divided into four classes: astrocytes, oligodendrocytes, ependymal cells and microligal cells. Astrocytes descend from a primitive neuroepithelial stem cell line within the ependymal zone. The exact function of astrocytes is unknown. Astrocytes probably provide support for the delicate neurons, contribute to the synthesis and degradation of neurotransmitters, control the ionic environment of the neurons and provide spacing between neurons.
Glial fibrillary acidic protein (GFAP) is expressed primarily in astrocytes of the central nervous system (including Mueller cells of the retina and non-myelinating Schwann cells of the peripheral nervous system). GFAP is a 50 kD intracytoplasmic protein that is the primary intermediate filament in the cytoskeleton of astrocytes. Mouse and human GFAP genomic genes have been cloned and sequenced as described in U.S. Pat. No. 5,267,047, incorporated herein by reference. The GFAP gene contains a basal promoter consisting of a TATA box and a CAAT box. Several enhancer and silencer sequences have also been identified. The enhancers for GFAP expression are found between xe2x88x92250 and xe2x88x9280 bp and between xe2x88x921980 and xe2x88x921500 bp. These positive control regions contain consensus sequences for many transcription factors including a cAMP response element and binding sites for the Sp-1, NF-1, AP-1 and AP-2 transcription factors. Tissue specificity is conferred by a human GFAP consensus sequence (hgcs) located in the xe2x88x921980 to xe2x88x921500 bp region. The transactivating protein which binds to this site has not been identified.
Reactive gliosis (also known as astrogliosis) occurs in response to almost any insult, physical or chemical, to the central nervous system (CNS). Reactive gliosis is characterized by hypertrophy of the astrocyte cell body and its processes, accompanied by an increase in expression of GFAP. One of the major problems in neurotoxicity screening is the diversity of insults that are to be tested and the highly specific nature of their targets, particularly for the pharmacological agents that may affect very discrete populations of neurons. Reactive gliosis in glial cells, in which up-regulation of GFAP is an invariant part, represents a robust change in the central nervous system following injuries to all of the relevant cell types in the central nervous system (neurons, oligodendrocytes, vascular elements, and astrocytes themselves). In the peripheral nervous system, a similar increase in GFAP occurs following both traumatic and toxic injuries tp peripheral nerve [(Mancardi et al., J.Neurosci. 102, 177 (1991); Toews et al., J.Neurosci. 12, 3676 (1992); Quattrini et al., Glia 17, 294 (1996)]. In frogs, peri-synaptic Schwann cells at the neuromuscular junction also respond to degeneration of the nerve terminals by forming sprouts and increasing expression of GFAP.
The correlation between the upregulation of GFAP expression and neural injury has been recognized as providing a possible biochemical indicator of neurotoxic or physical damage to the CNS. (See Mucke, The New Biologist, Vol. 3, No. 5, 465 (May 1991); O""Callaghan, Neurotoxicology and Teratology, Vol. 13, 275 (1991); O""Callaghan, Psychopharmacology Bulletin 30, 549 (1994); Verderber et al., Invest. Ophthalmol. Visual Sci. 36, 1137 (1995); and Wu et al., J.Neurosci. Res. 51, 675 (1998).) Mice transgenic for a GFAP-lacZ transgene exhibit increased production of the fusion protein in astrocytes of physically damaged brain and retina neural tissue. Likewise, exposure of mice to chemical neurotoxins results in increased wild-type GFAP expression as measured by immunohistochemistry and ELISA assays. Reactive gliosis in response to neurotoxin challenge is dose-, time-, and region-dependent. ELISA assays indicate that gliosis occurs at toxicant levels below those that cause light microscopic evidence of cell loss or damage.
The green fluorescent protein (GFP), a single peptide of 238 amino acids derived from the jellyfish Aequorea victoria, absorbs blue light and emits green light without a requirement for any cofactor or substrate. After the formation of its fluorophore by endogenous posttranslational cyclization, GFP is quite stable and remains fluorescent even after the harsh treatments found in many biochemical assays, such as 1% sodium dodecyl sulphate (SDS), 4% formaldehyde, and incubation at 65xc2x0 C. Since the first report of its use in Escherichia coli and Caenorhabditis elegans by Chalfie et al., Science 263, 802 (1994), GFP has found many applications as a reporter gene in a number of higher organisms including Drosophila [Wang et al., Nature 369, 400 (1994)] and zebrafish [Amsterdam et al., Dev. Biol. 171, 123 (1995); Peters et al., Dev. Biol. 171, 252 (1995)].
The versatility of the GFP is enhanced by its ability to remain fluorescent as a fusion protein allowing studies of the subcellular distribution and dynamics of various proteins, including NMDA receptors [Marshall et al., Neuron 14, 211 (1995); Niswender et al., J. Microsc. 180, 109 (1995); Aoki et al., FEBS Lett. 384, 193 (1996)]. Recently, a xe2x80x9chumanizedxe2x80x9d version of GFP has become available in which silent mutations were introduced to alter the codons to those more commonly used in mammals. The xe2x80x9chumanizedxe2x80x9d GFP is generally expressed at higher levels in mammalian systems than wild-type GFP. Mutant forms of GFP have become available which emit light of greater intensity or which exhibit wavelength shifts. (See Clontech Catalogue, 1998).
These genetically altered proteins offer increased sensitivity in assays for measuring neural insult. They offer an opportunity to assess the toxicity of substances at much lower levels than heretofore possible with conventional approaches. For example, WO94/17208 discloses a method of assessing toxicity by independent measurement of the expression of four different types of stress promoters. Detection of increased levels of stress gene expression is effected either by nucleic acid hybridization or a reporter gene such as the genes encoding glutathione transferase, luciferase, chloramphenicol acetyl transferase, or galactose kinase.
Another conventional approach is the use of cell cultures in studying gliosis, as reviewed recently by Wu, et al., supra. Astrocytes were cultured or co-cultured with other cell types under a variety of conditions to establish a baseline under one or more biochemical or morphological parameters, and then the baselines compared to cells subjected to various damaging sources of stress. Glial markers include GFAP, vimentin and trophic factor.
Over the past several years considerable effort has gone into the development of non-invasive imaging techniques for studies of tissue structure, metabolism, and most recently, gene expression. Non-invasive imaging in neurotoxicity screening would offer particular benefits in that testing would not require sacrifice of the animal, thereby reducing costs and improving animal welfare. Scientifically, a major advantage would be the possibility of repeat measurements on the same animal over time, to assess longer term effects of potentially toxic substances.
Accordingly, it is an object of the present invention to provide an assay system free of the artifacts of tissue culture. It is also an object of the present invention to provide assays for physical and neurotoxic challenges to the nervous system, more sensitive to low dose toxicants than conventional methods. It is a still further object to provide a non-invasive assay of neurologic toxicity capable of monitoring toxic effects over a period of time on more than one occasion in the same animal.
The present invention provides a rodent, preferably a mouse, which expresses a transgene encoding a humanized fluorescent green protein gene operably linked to a glial fibrillary acidic protein promoter. In this mouse the fluorescent green protein is upregulated specifically in glial cells such as astrocytes, Schwann cells, and Mueller cells in response to neural insult of a chemical or physical nature in which neural degeneration is manifest. The usefulness of the mouse lies in the ability to assay upregulation of the fluorescent green gene by visualizing fluorescence with a confocal microscope directly from the retina or cornea. The retinal site is a non-invasive locus for study of systemic toxicity. The cornea is particularly well suited to assessing toxicity of substances applied directly to an organ containing glial cells without invading the body.
The mouse is engineered by insertion of a genetic construct into the pronucleus (preferably the male pronucleus) of a mammalian zygote, and allowing stable genomic integration to occur naturally. The zygote is then transferred to a receptive uterus, and allowed to develop to term. While the mouse is a preferred species, rats and rabbits are also potential candidates for pronuclear insertion. The genetic construct which renders the zygote transgenic comprises a full length glial fibrillary acidic protein promoter to provide glial cell specific expression. The promoter is located 5xe2x80x2 of and operably linked to a mutant gene encoding fluorescent green protein, and a segment of DNA located 3xe2x80x2 of the mutant fluorescent green protein encoding gene containing signal sequences for proper RNA splicing and polyadenylation.
More specifically, the genetic construct contains DNA sequences in 5xe2x80x2 to 3xe2x80x2 order as follows: a glial fibrillary acidic protein promoter having at its 3xe2x80x2 end a sequence corresponding to SEQ ID NO: 1, a fluorescent reporter gene operably linked 3xe2x80x2 thereto, and polyadenylation signal sequence linked 3xe2x80x2 of the reporter gene. The fluorescent reporter gene is preferably hGFP-S65T green fluorescent protein gene, EGFP-1 green fluorescent gene, or EYFP-1 green fluorescent protein gene, or any variant thereof having mammalian compatible or humanized sequences (e.g. codon modification which renders the construct more compatible with mammalian ribosome translation) and a mutation increasing its light emission coefficient.
In preferred embodiments, the genetic construct of the present invention has at the 3xe2x80x2 end of the hGFP-S65T gene a joining sequence (joining the green gene and the polyadenylation signal containing sequence) corresponding to SEQ ID NO: 2 and has at its 5xe2x80x2 end a sequence (joining the promoter to the reporter gene) a bridging sequence corresponding to SEQ ID NO: 3.
The genetic construct is further characterized in having junctions between the 5xe2x80x2 to 3xe2x80x2 array of genetic elements, comprising a first junctional nucleic acid sequence at the 3xe2x80x2 end of the glial fibrillary acidic protein promoter linked to a second junctional sequence at the 5xe2x80x2 end of the humanized fluorescent reporter gene, together with a third junctional sequence at the 3xe2x80x2 end of the humanized fluorescent reporter gene intersecting and linked to a fourth junctional sequence at the 5xe2x80x2 end of a polyadenylation sequence, wherein the first junctional sequence is contiguous with the second junction sequence, and the third and fourth junctional sequences are thereby contiguous to each other.
In the method of the present invention, a mouse is provided which expresses the constructs disclosed hereinabove, exposing the mouse to a substance suspected of neurotoxicity and visualizing the presence of green fluorescence signal in glial cells such as astrocytes, Mueller cells, or Schwann cells to confirm upregulation of the glial fibrillary acidic protein promoter responsive to cellular degeneration associated with chemical or physical insult. In a quantitative assay of great sensitivity, the fluorescence signal in a predetermined area of exposure and visualization is calculated as the average pixel intensity for the area, and then the fluorescence signal is compared to a control fluorescence signal obtained from a control transgenic mouse not exposed to the target or test substance.
Confocal microscopy of the retinal Mueller cells of a live mouse or the Schwann cells of the cornea can be monitored by training the laser beam onto the desired region, and detecting the level of green fluorescence emitted. In this way the above objects of the invention may be realized in obtained sensitive toxicological data of either systemically or topically administered substances without invasive procedures. The assay can be performed sequentially many times on the same animal.